ABSTRACT
Pyrin domain-only proteins (POPs) are recently evolved, primate-specific proteins demonstrated in vitro as negative regulators of inflammatory responses. However, their in vivo function is not understood. Of the four known POPs, only POP2 is reported to regulate NF-κB-dependent transcription and multiple inflammasomes. Here we use a transgenic mouse-expressing POP2 controlled by its endogenous human promotor to study the immunological functions of POP2. Despite having significantly reduced inflammatory cytokine responses to LPS and bacterial infection, POP2 transgenic mice are more resistant to bacterial infection than wild-type mice. In a pulmonary tularaemia model, POP2 enhances IFN-γ production, modulates neutrophil numbers, improves macrophage functions, increases bacterial control and diminishes lung pathology. Thus, unlike other POPs thought to diminish innate protection, POP2 reduces detrimental inflammation while preserving and enhancing protective immunity. Our findings suggest that POP2 acts as a high-order regulator balancing cellular function and inflammation with broad implications for inflammation-associated diseases and therapeutic intervention.
Subject(s)
Francisella/immunology , Gram-Negative Bacterial Infections/immunology , Gram-Negative Bacterial Infections/prevention & control , Inflammation/immunology , Tularemia/immunology , Tularemia/prevention & control , Animals , Cell Line , Female , HEK293 Cells , HeLa Cells , Humans , Inflammasomes/immunology , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Listeria monocytogenes/immunology , Macrophages/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , NLR Family, Pyrin Domain-Containing 3 Protein/biosynthesis , Neutrophils/immunology , Promoter Regions, Genetic/genetics , Salmonella typhimurium/immunology , Transcription Factor RelA/biosynthesis , Tularemia/microbiology , U937 CellsABSTRACT
When resectable, invasive pancreatic ductal adenocarcinoma (PDAC) is most commonly treated with surgery and radiochemotherapy. Given the intricate local anatomy and locoregional mode of dissemination, achieving clean surgical margins can be a significant challenge. On the basis of observations that cathepsin E (CTSE) is overexpressed in PDAC and that an United States Food and Drug Administration (FDA)-approved protease inhibitor has high affinity for CTSE, we have developed a CTSE optical imaging agent [ritonavir tetramethyl-BODIPY (RIT-TMB)] for potential intraoperative use. We show nanomolar affinity [half maximal inhibitory concentration (IC50) of 39.9 ± 1.2 nM] against CTSE of the RIT-TMB in biochemical assays and intracellular accumulation and target-to-background ratios that allow specific delineation of individual cancer cells. This approach should be useful for more refined surgical staging, planning, and resection with curative intent.
Subject(s)
Boron Compounds , Carcinoma, Pancreatic Ductal/metabolism , Cathepsin E/metabolism , Fluorescent Dyes , HIV Protease Inhibitors , Pancreatic Neoplasms/metabolism , Ritonavir , Animals , Biopsy , Carcinoma, Pancreatic Ductal/diagnosis , Carcinoma, Pancreatic Ductal/pathology , Cathepsin E/antagonists & inhibitors , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , HIV Protease Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Mice , Pancreatic Neoplasms/diagnosis , Pancreatic Neoplasms/pathology , Ritonavir/analogs & derivatives , Ritonavir/pharmacologyABSTRACT
Observing drug responses in the tumor microenvironment in vivo can be technically challenging. As a result, cellular responses to molecularly targeted cancer drugs are often studied in cell culture, which does not accurately represent the behavior of cancer cells growing in vivo. Using high-resolution microscopy and fluorescently labeled genetic reporters for apoptosis, we developed an approach to visualize drug-induced cell death at single-cell resolution in vivo. Stable expression of the mitochondrial intermembrane protein IMS-RP was established in human breast and pancreatic cancer cells. Image analysis was then used to quantify release of IMS-RP into the cytoplasm upon apoptosis and irreversible mitochondrial permeabilization. Both breast and pancreatic cancer cells showed higher basal apoptotic rates in vivo than in culture. To study drug-induced apoptosis, we exposed tumor cells to navitoclax (ABT-263), an inhibitor of Bcl-2, Bcl-xL, and Bcl-w, both in vitro and in vivo. Although the tumors responded to Bcl-2 inhibition in vivo, inducing apoptosis in around 20% of cancer cells, the observed response was much higher in cell culture. Together, our findings show an imaging technique that can be used to directly visualize cell death within the tumor microenvironment in response to drug treatment.
Subject(s)
Aniline Compounds/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis , Breast Neoplasms/physiopathology , Mitochondrial Membranes/physiology , Molecular Imaging/methods , Pancreatic Neoplasms/physiopathology , Sulfonamides/pharmacology , Animals , Breast Neoplasms/drug therapy , Cell Line, Tumor , Female , Green Fluorescent Proteins , Humans , Image Processing, Computer-Assisted , Mice , Mice, Nude , Mitochondrial Membranes/drug effects , Mitochondrial Proteins/metabolism , Pancreatic Neoplasms/drug therapy , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Recombinant Fusion Proteins/metabolism , Single-Cell Analysis , Tumor MicroenvironmentSubject(s)
Enzyme Inhibitors/chemical synthesis , Poly(ADP-ribose) Polymerase Inhibitors , Radiopharmaceuticals/chemical synthesis , Animals , Cell Line, Tumor , Cyclooctanes/chemistry , Enzyme Inhibitors/chemistry , Fluorine Radioisotopes/chemistry , Humans , Mice , Mice, Nude , Neoplasms/diagnosis , Phthalazines/chemistry , Piperazines/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Positron-Emission Tomography , Radiopharmaceuticals/chemistrySubject(s)
Green Fluorescent Proteins/metabolism , Microscopy, Fluorescence/methods , Phthalazines/chemistry , Piperazines/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Cell Line, Tumor , Cell Nucleus/metabolism , Cytoplasm/metabolism , Green Fluorescent Proteins/genetics , Humans , Kinetics , Ligands , Molecular Structure , Phthalazines/pharmacology , Piperazines/pharmacology , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/genetics , Protein Transport/drug effects , Transfection , Xanthenes/chemistryABSTRACT
Inhibition of focal adhesion kinase (FAK) delays transendothelial migration of breast cancer cells. Here we investigate whether phosphorylation of specific tyrosine residues of FAK (397, 861, and 925) known to control aspects of cell migration on extracellular matrix (ECM), are also involved in transendothelial migration. AU-565 and MDA-MB-231 cells expressing Phe397 FAK show delayed or decreased transendothelial migration, demonstrating the involvement of the FAK autophosphorylation site. Only MDA-MB-231 cells expressing Phe861 FAK exhibit delayed transendothelial migration. Neither MDA-MB-231 nor AU-565 cells expressing Phe925 FAK show a change in transendothelial migration compared to untreated cancer cells. These findings suggest that modified signaling mechanisms regulate cancer cell migration through an endothelial monolayer versus those involved in cell migration on or through ECM.
Subject(s)
Breast Neoplasms/physiopathology , Breast Neoplasms/secondary , Endothelium, Vascular/physiopathology , Extracellular Matrix/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Breast Neoplasms/pathology , Cell Adhesion , Cell Line, Tumor , Cell Movement , Endothelium, Vascular/pathology , Humans , PhosphorylationABSTRACT
We used terahertz differential time-domain spectroscopy (THz-DTDS) to measure minute changes of bovine lung microvessel endothelial cells (BLMVEC) in response to vascular endothelial growth factor (VEGF). These changes were reflected by alterations in THz wave attenuations and THz dielectric properties of the treated cells. The VEGF-induced THz attenuations of cell monolayers correlated well with changes in transendothelial resistance, as measured using electric cell-substrate impedance sensing (ECIS). However, the morphological differences that gave rise to these changes were not observed with standard optical phase contrast microscopy. We conclude that THz-DTDS is a highly sensitive, non-invasive, powerful new tool to measure minute changes in the morphology of live, cultured cell monolayers. This method enables spectroscopic investigations of cells in the THz band, providing information unavailable through other conventional methods such as optical phase contrast microscopy and ECIS.
Subject(s)
Cell Culture Techniques/instrumentation , Endothelial Cells/drug effects , Endothelial Cells/physiology , Microwaves , Spectrum Analysis/methods , Vascular Endothelial Growth Factor A/administration & dosage , Animals , Cattle , Cell Culture Techniques/methods , Cell Size/drug effects , Cells, Cultured , Equipment Design , Equipment Failure AnalysisABSTRACT
Transendothelial migration of cancer cells from the vasculature into tissue stroma is a final step in the metastatic cascade, prior to formation of secondary tumors. Due to its role in 2-dimensional migration of cells on extracellular matrix proteins, we hypothesized that focal adhesion kinase (FAK) promotes transendothelial migration of cancer cells. AU-565 cells are weakly invasive metastatic breast adenocarcinoma cells that migrate through bovine lung microvessel endothelial cell monolayers. Electric cell-substrate impedance sensing detects a significant decrease in monolayer resistance upon addition of AU-565 cells. Immunofluorescence microscopy and filter-based migration assays demonstrate that this drop in resistance correlates with transendothelial migration. Transfection of AU-565 cells with FAK siRNA results in significantly diminished transendothelial migration of AU-565 cells within 15h. Expression of the dominant negative FAK inhibitor FAK-related non-kinase (FRNK) also results in delayed AU-565 transendothelial migration, whereas over-expression of wildtype FAK does not impact transendothelial migration substantially. These results demonstrate that FAK affects the rate of a key step in the metastatic cascade.
